Feature Articles

PTMs Progress Toward Designer Proteins

Advances in the Analysis and Biosynthesis of Glycans Are Transforming the Field

Designer Proteins?

Just as humanized antibodies seek to duplicate the amino acid sequence produced in humans, researchers are hot on the heels of “designer” glycoproteins with tailor-made glycans. Since glycosylation cannot be genetically encoded, investigators must resort to artificial means to achieve desirable, glycosylation patterns. “Glycan trimming,” a method employed by Heather Desaire, Ph.D., a chemistry professor at the University of Kansas (Lawrence), may be one step toward glycosylation-by-design.

“The method is designed so that either an individual monosaccharide type is removed, or, if more than one deglycosylating enzyme is used, multiple monosaccharide types could be trimmed from the glycoprotein,” Dr. Desaire says. After the enzymatic reactions, purification and analysis ensures the desired glycosylation profile was obtained, and that the protein folded properly.

Structure-activity relationships for glycosylation are still in an early stage. Most correlations are empirical. Given that limitation, glycan trimming may be employed to improve circulating half-life, in the case of mannose-trimming, by reducing the opportunity for mannose binding lectin (MBL) to clear glycoproteins from circulation.

“We have not shown experimentally that trimmed proteins have longer circulation lifetimes, but the binding of MBL to high mannose glycoproteins is well known,” comments Dr. Desaire. Other potential benefits are reducing immunogenicity by removing sialic acid and other immunogenic residues, and improving stability or solubility.

For years protein glycosylation was believed to be an exclusive trait of eukaryotes. The discovery, in 1999, that the bacterium Campylobacter jejuni added glycans to proteins, was therefore a startling revelation. Research by Christine Syzmanski (National Research Council of Canada), and Brendan Wren and Rebecca Langdon, Ph.D., a scientist at the London School of Hygiene and Tropical Medicine, among others, has firmly established that bacteria conduct widespread N-glycosylation of proteins.

Dr. Langdon’s group has identified genes implicated in protein glycosylation in several different Campylobacter and Helicobacter species. “While we now have a good understanding of how N-glycosylation functions in C. jejuni, we are still unclear of its role in the biology of the bacterium and also how the system works in the other bacteria where the genes have been identified,” Dr. Langdon says.

Using a technique known as protein-glycan-coupling technology, she has expressed protein-glycosylation genes in E. coli, an organism more familiar to bioprocessors than Campylobacter or Helicobacter. Dr. Langdon is currently developing this system to produce protein-glycoconjugate vaccines.

Other groups, particularly Professor Mario Feldman’s at the University of Alberta are working on O-glycosylation as well. However, with O-glycosylation there is less control over the protein acceptor site. “Using N-glycosylation, we can engineer proteins to include the acceptor site for attachment of the sugar substrate. This creates the opportunity to theoretically link any polysaccharide to any protein.”

The implications for (future) glycoprotein manufacture in simple bacterial systems are tremendous, but for now the near-term potential remains in glycoconjugate vaccines. Dr. Langdon has been collaborating with GlycoVaxyn, to commercialize such vaccines.

PTM-Based Medicine

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The vicious cycle of immune activation, antibody production, and inflammation can be inhibited by SM101, according to SuppreMol.

PTM is most often associated with glycosylation, but numerous other modifications occur post-translation. Among the most significant is the formation of disulfide bonds that are critical for protein folding. Researchers at SuppreMol are using a type of chemical oxidation to form disulfide bonds in SM101, an early clinical-stage drug for autoimmune diseases.

SM101 is a recombinant, soluble, non-glycosylated version of FcγRIIb, a human Fcγ receptor that binds to autoantibody/autoantigen complexes and blocks Fcγ receptors on immune cells. When FcγRIIb is working properly the immune response is attenuated, and the inflammation cascade typically of autoimmune diseases is prevented.

Patients with certain autoimmune diseases show levels of soluble Fc receptors in their blood that are significantly lower than in healthy individuals. SM101 essentially supplements these under-represented soluble receptors. The absence of glycans allows SuppreMol to manufacture the drug in E. coli instead of cell culture.

Under a European orphan drug designation, SuppreMol is targeting SM101 to idiopathic thrombocytopenic purpura, an autoimmune disease that depletes platelets (resulting in spontaneous, sometimes life-threatening bleeding), and causes the spleen to swell. The drug has successfully passed through early Phase I; a Phase Ia/IIb patient study is expected to begin in early 2010. Lupus and rheumatoid arthritis are other potential diseases treatable with SM101 that will be addressed at a later stage of development.

Current treatments involve knocking down the immune system or stimulating platelet formation. “But making more platelets also leads to an increase in auto-antigens,” notes Peter Sondermann, Ph.D., CSO. “The right strategy probably involves balancing the immune system against platelet formation. We believe we can modulate the immune system in a way that platelets are not regarded as an antigen, at least for a distinct time.”

The PTM angle here is Dr. Sondermann’s technique for forming disulfide bridges within SM101 after it is released from inclusion bodies in E. coli. During the preclinical and early clinical development phase, this was accomplished using oxidized glutathione in the presence of arginine that supports the folding, but both substances are expensive and must be produced in microorganisms.

“Because we’re making a drug with it, you have to track where the material comes from,” Dr. Sondermann says. The synthetic disulfide-bridge mediating molecule cystamine and the folding-assistant urea are now used in the process. Both compounds are inexpensive, widely available, and reduce safety concerns.

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